4718 | Chem. Commun., 2016, 52, 4718--4721 This journal is©The Royal Society of Chemistry 2016

Cite this:Chem. Commun., 2016,

52, 4718

A fluorescent surrogate of thymidine in duplexDNA†

Guillaume Mata, Olivia P. Schmidt and Nathan W. Luedtke*

DMAT is a new fluorescent thymidine mimic composed of 20-deoxy-

uridine fused to dimethylaniline. DMAT exhibits the same pKa and

base pairing characteristics as native thymidine residues, and its

fluorescence properties are highly sensitive to nucleobase ionization,

base pairing and metal binding.

Nucleobase analogs constitute an important family of fluores-cent probes.1 They can be positioned in nucleic acid structureswith high precision, and their photophysical properties arehighly sensitive to local polarity,2 viscosity,3 and pH.4 Thesefeatures facilitate specific monitoring of biochemical trans-formations,5 conformational changes,6 metal binding,7 and basepairing interactions.8

Nucleobase ionization can mediate proton-coupled folding,9

metal binding,10 and/or the catalytic activities of certain nucleicacids at neutral pH.11 Thymidine (T) and uracil (U) are amongthe most inherently acidic residues,12 but fluorescent analogscapable of reporting pyrimidine ionization in nucleic acids arescarce. Previously reported examples utilized biaryl or triarylfluorophores,4c,d having unreported or highly perturbed pKa

values (pKa E 6.8) as compared to unmodified T and U residues(pKa E 9.5).12 Here we report ‘‘N,N-dimethylaniline-20-deoxy-thymidine’’ or ‘‘DMAT’’ that exhibits the same pKa and basepairing characteristics as thymidine, as well as fluorescenceproperties that can be used to monitor nucleobase ionization,base pairing and metal binding reactions in DNA.

DMAT was designed to have the same Watson–Crick face andpKa as thymidine. To generate a push–pull fluorophore, anelectron donating group was incorporated at the C6 position ofa quinazoline core.9,13 This position was selected because it isnot in conjugation with N3–H, and therefore expected to havelittle or no impact on its acidity. Molecular orbital calculationspredicted charge transfer from a dimethylaniline-centeredHOMO to a pyrimidine-centered LUMO with a HOMO–LUMO

energy gap (DE) = 2.44 eV (Fig. 1). DMAT was therefore predictedto be a ‘‘push–pull’’ fluorophore in its neutral form. In contrast,the DMAT anion has a pyrimidine-centered HOMO, and a largerDE = 2.81 eV. We therefore expected a blue-shift in DMATfluorescence upon its deprotonation.

The synthesis of DMAT (1) commenced from the previously-reported nucleoside 2 (Scheme 1).14 Buchwald–Hartwig couplingwith Me2NH gave the known compound 3 in 82% yield.9 Additionof fluoride ions to 3 afforded the new nucleoside DMAT (1) in a

Fig. 1 Structure of the DMAT nucleoside (A), and its conjugate base (B).HOMOs, LUMOs, and their relative energies were calculated from DFT-optimized geometries using LSDA/pBP86/DN**.

Scheme 1 Synthesis of DMAT (1). Reaction conditions: (a) Me2NH, Pd2(dba)3(5 mol%), JohnPhos (20 mol%), KOtBu, THF, dioxane, 60 1C, 2 h, 82% yield.(b) TBAF, THF, 23 1C, 2 h, 62% yield.

Department of Chemistry, University of Zurich, Winterthurerstrasse 190,

CH-8057 Zurich, Switzerland. E-mail: nathan.luedtke@chem.uzh.ch

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc09552b

Received 18th November 2015,Accepted 25th February 2016

DOI: 10.1039/c5cc09552b

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62% yield (see ESI† for synthetic details and characterization).1H–1H ROESY cross-peaks were observed between H8–H50, H8–H30,and H8–H20b of DMAT (1), indicating an anti-conformation ofthe nucleobase (Fig. 2). Cross-peaks were also observed betweenH10–H40, which confirmed the b-stereochemistry of the anomericposition.14 DMAT (1) therefore possesses the same glycosidic bondconformation and stereochemistry as 20-deoxythymidine.

Under neutral conditions, DMAT (1) exhibits an exceptionallylarge Stoke’s shift, with an absorbance maximum (labs) = 357 nmand emission maximum (lem) = 522 nm (Table 1). To character-ize its environmental sensitivity, the labs and lem of DMAT (1)were measured in various water/dioxane mixtures (Table S2 andFig. S1, ESI†). A linear correlation (R2 = 0.980) with a large slopeof 177 cm�1 kcal�1 mol�1 was obtained by plotting the Stoke’sshift of DMAT (1) against Reichardt’s solvent polarity parameter(E30

T ).15,16 Together these results confirm that DMAT (1) is apush–pull fluorophore. Interestingly, DMAT (1) exhibits a two-fold higher quantum yield in D2O (f = 0.07) than H2O (f = 0.03,Table 1), suggesting that proton transfer with bulk solvent providesan effective nonradiative decay pathway.7a

To evaluate the fluorescence sensitivity of DMAT (1) towardsnucleobase ionization, its absorbance and emission spectra wererecorded at different pH values. Consistent with DFT calculations,the emission maximum of DMAT (1) shifted towards the blue withincreasing pH (Fig. 3A). This was accompanied by a dramaticincrease in fluorescence intensity. The absorbance and fluorescencechanges were plotted against pH to determine a pKa = 9.5 � 0.1,Fig. 3B. This value corresponds to the pKa of thymidine and uracil.12

To facilitate the site-specific incorporation of DMAT intoDNA, phosphoramidite 5 was prepared in two steps by standard

DMT-protection and phosphitylation reactions (Scheme 2).Using automated DNA synthesis, DMAT was incorporated at oneof four positions within the same, 21-residue DNA sequence(Table S3, ESI†). Three positions near the middle of the sequence(X13, X14 and X15) and a single position near the 50 terminus(X2) were selected in order to evaluate the impacts of vari-able flanking sequences and DNA end ‘‘breathing’’ motions,respectively.‡ The identity and purity of the purified oligo-nucleotides were confirmed using analytical HPLC and HR-MS(Table S4 and Fig. S2, ESI†).

Circular dichroism (CD) and thermal denaturation experi-ments were used to assess the impact of DMAT on the globalstructure and stability of duplex DNA. DMAT-containing duplexeswere prepared by heating and slow cooling with 1.1 equiv. of thecomplementary strand to give CD spectra consistent with theformation of B-form helices (Fig. S3, ESI†).18 CD spectra weremonitored as a function of temperature (Fig. S4, ESI†) to deter-mine the melting temperature of each duplex (Tm, Table 2).DMAT–A-containing duplexes exhibited nearly identical Tm valuesas the corresponding wild-type duplexes containing T–A basepairs (DTm = �0.2 to �1.7 1C). In contrast, duplexes containinga single C–A, A–A, DMAT–T or T–T mismatch at positionsX13–X15 caused a large loss in thermal stability (DTm = �4.8to �7.9 1C). End breathing motions of duplex DNA explain therelatively small changes when the mismatches were positionedat X2 (Table 2).

The fluorescence properties of DMAT were highly sensitive tothe global structure of the DNA containing it (Fig. S5, ESI,† andTable 3). At all three internal positions X13–X15, DMAT exhibiteda two-fold higher quantum yield and blue-shifted lem in duplex

Fig. 2 (A) Partial 1H–1H 2D ROESY of DMAT (1) in DMSO-d6. See ESI† forcomplete spectrum. (B) Energy-minimized conformation of DMAT accordingto DFT calculations (B3LYP/6-311G*). 1H–1H distances are shown in Å.

Table 1 Photophysical data of DMAT (1) and its conjugate base

Solvent labsa lem

b e(260)c e(labs)

c fd

H2O 357 522 15.0 2.9 0.03pH = 11.0 345 480 17.1 3.9 0.09D2O 355 520 13.0 2.7 0.07pD = 11.0 345 480 14.3 3.4 0.19

a Absorbance maxima (labs) in nm. b Emission maxima (lem) in nm.c Extinction coefficients (e) in 103 M�1 cm�1 were measured at 260 nmand at labs.

d Quantum yields (f) were calculated using quinine hemi-sulfate in 0.5 M H2SO4 as a fluorescent standard.17 Reproducibility iswithin �10% of each reported f value.

Fig. 3 (A) Absorbance (---) and emission (—) spectra (lex = 360 nm) of40 mM DMAT (1) in phosphate citric acid buffer (200 mM of Na2HPO4,100 mM of citric acid and 100 mM NaCl). (B) Single-wavelength analyses ofthe pH-dependent absorbance (400 nm, blue dots) and emission (470 nm,red dots) of DMAT (1).

Scheme 2 Synthesis of DMAT phosphoramidite (5). Reaction conditions:(a) DMT-Cl, pyridine, 23 1C, 45 min, 79% yield. (b) 2-Cyanoethyl-N,N-diisopropyl-chlorophosphoramidite, DIPEA, CH2Cl2, 0 1C to 23 1C, 45 min,86% yield. See ESI† for synthetic details and characterizations.

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4720 | Chem. Commun., 2016, 52, 4718--4721 This journal is©The Royal Society of Chemistry 2016

versus single-stranded DNA. Decreased probe hydration uponduplex formation is probably responsible for these differences,because the DMAT nucleoside (1) exhibited higher quantum yieldsand blue-shifted lem in organic versus aqueous solvents (Table S2and Fig. S1, ESI†). At all four positions of incorporation, higherfluorescence anisotropy was observed in duplex DNA (r = 0.09–0.18)as compared to unfolded structures (r = 0.03–0.06), consistentwith large losses in dynamic motions of the probe upon duplexformation (Table 3).

The photophysical properties of DMAT were sensitive tomatched versus mismatched base pairing in duplex DNA. DMATexhibited a higher quantum yield and blue-shifted labs andlem in DMAT–A base pairs as compared to DMAT–T, DMAT–G andDMAT–C mismatches (Table 4). Changes in probe hydrationand base stacking are likely responsible for these differences,because similar trends were also observed when comparingduplex versus single-stranded DNA containing DMAT (Table 3).Taken together with the thermal denaturation results (Table 2),these data provide additional evidence that DMAT exhibits thesame base pairing specificity as T.

Metal-mediated base pairing interactions serve as importantrecognition motifs in biological and material sciences.10 Forexample, HgII ions specifically bind to opposing thymine residuesto form T–Hg–T base pairs19 that can cause miscoding of DNAsynthesis in vitro and possibly in vivo.20 To evaluate the ability ofDMAT–T to mimic T–T in duplex DNA, Tm values were measuredin the presence or absence of 1.0 equiv. of HgII (Table 5 andFig. S6–S8, ESI†). Only small increases in thermal stabilities(DTm = +0.8 to +1.5 1C) were observed when HgII was added to

duplexes containing a DMAT–T or T–T at position X2, whereasmuch larger increases were observed at positions X13–X15(DTm = +2.9 to +6.0 1C). At all four positions, the same Tm valueswere obtained for duplexes containing DMAT–Hg–T as T–Hg–T. Incontrast, the addition of 1.0 equiv. of HgII to duplexes containinga C–T mismatch resulted in no increase in thermal stability ascompared to the mismatch alone (Table 5). Taken together, theseresults demonstrate the excellent mimicry of DMAT for thymidineresidues in the demanding context of T–HgII–T base pairs.

The fluorescence properties of DMAT can be utilized to monitorsite-specific binding of HgII ions to DMAT–T sites. Bi-phasicfluorescence quenching was observed upon addition of HgII toduplex DNA containing a DMAT–T mismatch, giving a 95% decreasein fluorescence intensity upon addition of 3 equiv. of HgII (Fig. 4).Similar results were obtained when DMAT was located at all fourpositions X2, X13, X14 and X15 (Table S5 and Fig. S9, ESI†). Thebiphasic quenching (Fig. 4B) mirrors the increases in Tm valuesobtained when HgII is added to duplexes containing a T–T mis-match (Fig. S6, ESI†). A comparison of these results reveals that thesteep slopes observed between 0.0 to 1.0 equiv. of added HgII

are a result of T–T-specific binding, and the shallow slopes

Table 2 Thermal denaturation melting (Tm) temperatures (1C) of duplexesa

Position T–A DMAT–A C–A A–A DMAT–T T–T

X2 69.0 68.8 67.3 67.5 68.0 67.0X13 68.8 67.1 60.9 62.4 62.3 61.5X14 68.7 67.3 61.3 63.9 62.5 61.5X15 68.9 68.0 61.2 62.7 64.0 62.8

a All samples contained 5 mM of DNA in phosphate citric acid buffer(200 mM of Na2HPO4, 100 mM of citric acid, and 100 mM NaCl orNaNO3) at pH = 7.35. Reproducibility is within �0.3 1C of each reportedvalue. See Table S3, ESI for duplex sequences.

Table 3 Photophysical properties of DMAT in DNA (X = DMAT)

Structure Position labsa lem

b rc fd

Duplex X2 355 505 0.18 0.03X13 355 504 0.09 0.20X14 355 492 0.14 0.13X15 355 486 0.15 0.11

Unfolded X2 365 515 0.03 0.05X13 365 515 0.05 0.09X14 365 516 0.06 0.06X15 365 515 0.05 0.05

a Absorbance maxima (labs) in nm. b Emission maxima (lem) in nm.c Fluorescence anisotropy (r) with lex = 375 nm and lem = 500 nm.d Quantum yields (f) calculated using the DMAT nucleoside (f = 0.03) asa fluorescent standard. Reproducibility is within�30% of each reportedf value. All samples contained 4 mM of DNA in a buffer containing20 mM of Na2HPO4, 10 mM of citric acid and 10 mM NaCl (pH = 7.35).See Table S3, ESI for duplex sequences.

Table 4 Photophysical properties of DMAT in duplex DNAa

Position Pairing labs lem r f

X13 DMAT–A 355 504 0.09 0.20DMAT–T 365 505 0.09 0.13DMAT–G 365 508 0.10 0.09DMAT–C 365 498 0.10 0.12

X15 DMAT–A 355 486 0.15 0.11DMAT–T 365 500 0.12 0.08DMAT–G 370 505 0.12 0.06DMAT–C 370 502 0.17 0.05

a See Table 3 footnotes for experimental details and symbol definitions.

Table 5 Thermal denaturation melting (Tm) temperatures (1C) of duplexesa

Position T–T T–T + HgII DMAT–T DMAT–T + HgII C–T C–T + HgII

X2 67.0 68.5 68.0 68.8 66.8 66.3X13 61.5 67.5 62.3 67.1 60.4 61.1X14 61.5 65.1 62.5 65.7 60.4 60.3X15 62.8 65.7 64.0 66.9 60.6 60.9

a See Table 2 footnotes for experimental details.

Fig. 4 (A) Fluorescence spectra (lex = 260 nm) of duplex X13 in the absence(black) and in the presence of variable Hg(ClO4)2. (B) Plot of fluorescenceintensity (lem = 500 nm) versus equiv. of HgII ions. All samples contained 5 mMof DNA in phosphate citric acid buffer (200 mM of Na2HPO4, 100 mM of citricacid and 100 mM NaNO3) at pH = 7.35. See Table S3, ESI† for duplex sequences.

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between 2.0 to 3.0 equiv. are due to non-specific interactions. Incontrast to DMAT–T, very little fluorescence quenching (�20%)was observed upon the addition of HgII ions to duplex DNAcontaining a DMAT–A base pair, or a DMAT–G mismatch (Table S6and Fig. S10, ESI†). Conversely, the addition of ZnII, CuII, MgII,FeII, CaII, AgI, CdII, PdII and NiII ions to duplexes containing aDMAT–T mismatch resulted in little or no change in DMATfluorescence (Fig. S11, ESI†). These results are consistent withprevious studies demonstrating a high degree of specificity betweenT–T mismatches and HgII ions using thermal denaturation.19 Tothe best of our knowledge, our results provide the first example ofusing a fluorescent nucleobase analog to monitor a specific bindingreaction between DNA and HgII ions. DMAT will therefore enabledetailed kinetics analyses and large-scale screening efforts that arenot feasible using other analytical techniques.10d

A variety of fluorescent nucleoside analogs are available forsolid-phase synthesis of DNA and RNA.1–8 However, the vastmajority of these probes are quenched by their incorporationinto duplex nucleic acids, where they can disrupt duplexstability by as much as a base pair mismatch. Here we report anew fluorescent thymidine mimic composed of 20-deoxyuridinefused to dimethylaniline. According to thermal denaturation,fluorescence, and metal binding studies, DMAT exhibits the samebase pairing characteristics as native thymidine residues. Thequantum yield of DMAT (f = 0.03 in water) increases upon itsincorporation into duplex DNA (f = 0.11–0.20), where its fluores-cence properties are highly sensitive to nucleobase hydration,ionization, base pairing, and metal binding. Taken together,these results demonstrate that DMAT will enable a wide variety ofstudies aimed at characterizing biochemical transformations,conformational changes, site-specific metal binding, and basepairing interactions with single-nucleotide resolution.

Notes and references‡ DNA sequences: X2: CXC-TAA-CCC-TAA-CCC-TAA-CCC; X13: CCC-TAA-CCC-TAA-XCC-TAA-CCC; X14: CCC-TAA-CCC-TAA-CXC-TAA-CCC;X15: CCC-TAA-CCC-TAA-CCX-TAA-CCC; (X = DMAT, T (wt), C or A).

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